Genetic variation in DNA damage response pathway and response to Gemtuzumab Ozogamicin in pediatric AML: a report from the Children's Oncology Group.

PURPOSE: Comprehensive pharmacogenomics (PGx) evaluation of calicheamicin-pathway to identify predictive PGx markers of response to gemtuzumab ozogamicin (GO) treatment in acute myeloid leukemia (AML). PATIENTS AND METHODS: Single nucleotide polymorphisms (SNPs) in DNA-damage response (DDR) pathway genes were tested for association with event-free survival (EFS), overall-survival (OS), risk of relapse after induction 1 (RR1) in patients treated with standard chemotherapy consisting of Ara-C, Daunorubicin and Etoposide (ADE) with or without addition of GO on COG-AAML03P1 and COG-AAAML0531 trials (ADE+GO, n=755; ADE n=470). SNPs with significant association with any endpoint within ADE+GO arm but not in the ADE arm were tested using multi-SNP modeling to develop DDR_PGx7 Score. RESULTS: Patients with low-DDR_PGx7 score (<0) had significantly worse EFS (HR=1.51, 95%CI (1.21-1.89), P<0.001), worse OS (HR=1.59, 95%CI (1.22-2.08), P<0.001), and higher RR1 (HR=1.87, 95%CI(1.41-2.47), P<0.0001) compared to patients with high-DDR_PGx7 score (≥0) when treated with GO (ADE+GO cohort). However, no difference between low and high DDR_PGx7 score groups was observed for EFS, OS, and RR1 (all P>0.3) in patients treated on ADE arm. CONCLUSIONS: Our results suggest that DDR pathway-based pharmacogenomic score holds potential to predict outcome in patients treated with GO which consists of DNA damaging cytotoxin, calicheamicin. The potential clinical relevance for this score to personalize GO in AML requires further validation in independent and expanded cohorts.

ABCB1 SNP predicts outcome in patients with acute myeloid leukemia treated with Gemtuzumab ozogamicin: a report from Children's Oncology Group AAML0531 Trial.

Gemtuzumab-ozogamicin (GO), a humanized-anti-CD33 antibody linked with the toxin-calicheamicin-γ is a reemerging and promising drug for AML. Calicheamicin a key element of GO, induces DNA-damage and cell-death once the linked CD33-antibody facilitates its uptake. Calicheamicin efflux by the drug-transporter PgP-1 have been implicated in GO response thus in this study, we evaluated impact of ABCB1-SNPs on GO response. Genomic-DNA samples from 942 patients randomized to receive standard therapy with or without addition of GO (COG-AAML0531) were genotyped for ABCB1-SNPs. Our most interesting results show that for rs1045642, patients with minor-T-allele (CT/TT) had better outcome as compared to patients with CC genotype in GO-arm (Event-free survival-EFS: p = 0.022; and risk of relapse-RR, p = 0.007). In contrast, no difference between genotypes was observed for any of the clinical endpoints within No-GO arm (all p > 0.05). Consistent results were obtained when genotype groups were compared by GO and No-GO arms. The in vitro evaluation using HL60-cells further demonstrated consistent impact of rs1045642-T-allele on calicheamicin induced DNA-damage and cell-viability. Our results show the significance of ABCB1 SNPs on GO response in AML and warrants the need to investigate this in other cohorts. Once validated, ABCB1-SNPs in conjunction with CD33-SNPs can open up opportunities to personalize GO-therapy.

CD33_PGx6_Score Predicts Gemtuzumab Ozogamicin Response in Childhood Acute Myeloid Leukemia: A Report From the Children's Oncology Group.

PURPOSE: The US Food and Drug Administration recently announced reapproval of gemtuzumab ozogamicin (GO) for treatment of CD33-positive acute myeloid leukemia (AML), thus opening up opportunities to develop strategies for effective use of GO. In light of our recent report showing prognostic significance of CD33 splicing single nucleotide polymorphisms (SNPs), the objective of this study was to comprehensively evaluate CD33 SNPs for accurate prediction of patients with AML who are more or less likely to respond to GO. PATIENTS AND METHODS: We investigated the five new CD33 SNPs (rs2455069, rs35112940, rs61736475, rs1803254, and rs201074739) for association with CD33 leukemic cell surface expression and clinical response in pediatric patients with AML enrolled in the Children's Oncology Group AAML0531 trial. We further developed a composite CD33 pharmacogenetics (PGx) score using six CD33 SNPs (CD33_PGx6_score) for association with clinical outcome. RESULTS: Four CD33 SNPs were associated with cell surface CD33 levels and clinical response in the GO versus no-GO arms. Therefore, the CD33_PGx6_score was built using directional genotype scores for the previously reported splicing SNP and five new SNPs. Patients with a CD33_PGx6_score of 0 or higher had higher CD33 expression levels compared with patients with a score of less than 0 (P < .001). In addition, patients with a score of 0 or higher demonstrated an improved disease-free survival in the GO versus no-GO arms (62.5% ± 7.8% v 46.8% ± 8.3%, respectively; P = .008) and a reduced risk of relapse (28.3% ± 7.2% v 49.9% ± 8.4%, respectively; P < .001). No improvement from GO was observed in patients with a CD33-PGx6_score of less than 0. Consistent results were observed across the risk groups. CONCLUSION: In this study, we report a composite CD33_PGx6_score using directional genotype scores of CD33 SNPs. Once validated, our findings hold promise for use of the CD33_PGx6_score to guide efficient use of GO in patients with AML. In addition, because the CD33_PGx6_score considers SNPs with varying abundance in different ethnic groups, it has potential for global application.

CD33 Splicing Polymorphism Determines Gemtuzumab Ozogamicin Response in De Novo Acute Myeloid Leukemia: Report From Randomized Phase III Children's Oncology Group Trial AAML0531.

Purpose Gemtuzumab ozogamicin (GO), a CD33-targeted immunoconjugate, is a re-emerging therapy for acute myeloid leukemia (AML). CD33 single nucleotide polymorphism rs12459419 C>T in the splice enhancer region regulates the expression of an alternatively spliced CD33 isoform lacking exon2 (D2-CD33), thus eliminating the CD33 IgV domain, which is the antibody-binding site for GO, as well as diagnostic immunophenotypic panels. We aimed to determine the impact of the genotype of this splicing polymorphism in patients with AML treated with GO-containing chemotherapy. Patients and Methods CD33 splicing single nucleotide polymorphism was evaluated in newly diagnosed patients with AML randomly assigned to receive standard five-course chemotherapy alone (No-GO arm, n = 408) or chemotherapy with the addition of two doses of GO once during induction and once during intensification (GO arm, n = 408) as per the Children's Oncology Group AAML0531 trial. Results The rs12459419 genotype was CC in 415 patients (51%), CT in 316 patients (39%), and TT in 85 patients (10%), with a minor allele frequency of 30%. The T allele was significantly associated with higher levels of D2-CD33 transcript ( P < 1.0E-6) and with lower diagnostic leukemic cell surface CD33 intensity ( P < 1.0E-6). Patients with the CC genotype had significantly lower relapse risk in the GO arm than in the No-GO arm (26% v 49%; P < .001). However, in patients with the CT or TT genotype, exposure to GO did not influence relapse risk (39% v 40%; P = .85). Disease-free survival was higher in patients with the CC genotype in the GO arm than in the No-GO arm (65% v 46%, respectively; P = .004), but this benefit of GO addition was not seen in patients with the CT or TT genotype. Conclusion Our results suggest that patients with the CC genotype for rs12459419 have a substantial response to GO, making this a potential biomarker for the selection of patients with a likelihood of significant response to GO.

Genome-wide association analysis identified splicing single nucleotide polymorphism in CFLAR predictive of triptolide chemo-sensitivity.

BACKGROUND: Triptolide is a therapeutic diterpenoid derived from the Chinese herb Tripterygium wilfordii Hook f. Triptolide has been shown to induce apoptosis by activation of pro-apoptotic proteins, inhibiting NFkB and c-KIT pathways, suppressing the Jak2 transcription, activating MAPK8/JNK signaling and modulating the heat shock responses. RESULTS: In the present study, we used lymphoblast cell lines (LCLs) derived from 55 unrelated Caucasian subjects to identify genetic markers predictive of cellular sensitivity to triptolide using genome wide association study. Our results identified SNPs on chromosome 2 associated with triptolide IC50 (p < 0.0001). This region included biologically interesting genes as CFLAR, PPIl3, Caspase 8/10, NFkB and STAT6. Identification of a splicing-SNP rs10190751, which regulates CFLAR alternatively spliced isoforms predictive of the triptolide cytotoxicity suggests its role in triptolides action. Our results from functional studies in Panc-1 cell lines further demonstrate potential role of CFLAR in triptolide toxicity. Analysis of gene-expression with cytotoxicity identified JAK1 expression to be a significant predictor of triptolide sensitivity. CONCLUSIONS: Overall out results identified genetic factors associated with triptolide chemo-sensitivity thereby opening up opportunities to better understand its mechanism of action as well as utilize these biomarkers to predict therapeutic response in patients.

Cytochrome P450 2B6*5 Increases Relapse after Cyclophosphamide-Containing Conditioning and Autologous Transplantation for Lymphoma.

Cyclophosphamide (Cy) is a prodrug that depends on bioactivation by hepatic cytochrome P450 (CYP) enzymes for its cytotoxicity. We evaluated the influence of single nucleotide polymorphisms (SNPs) of CYP enzymes on the efficacy of autologous hematopoietic cell transplantation (HCT) for lymphoma. SNPs of 22 genes were analyzed in 93 patients with Hodgkin (n = 52) and non-Hodgkin lymphoma (n = 41) treated with high-dose Cy followed by autologous HCT between 2004 and 2012. Preparative regimens contained Cy (120 mg/kg) combined with carmustine/etoposide (n = 61) or Cy (6000 mg/m(2)) with total body irradiation (n = 32). Lack of complete remission as measured by pretransplant positron emission tomography was the sole clinical factor associated with increased risk of relapse (HR, 2.1). In genomic analysis, we identified a single SNP (rs3211371) in exon 9 (C > T) of the CYP2B6 gene (allele designation 2B6*5) that significantly impacted patient outcomes. After adjusting for disease status and conditioning regimen, patients with the CYP2B6*1/*5 genotype had a higher 2-year relapse rate (HR, 3.3; 95% CI, 1.6 to 6.5; P = .041) and decreased overall survival (HR, 13.5; 95% CI, 3.5 to 51.9; P = .008) than patients with the wild-type allele. Two-year progression-free survival for patients with 2 hypofunctional CYP2B6 variant genotypes (*5 and *6) was only 11% (95% CI, 1% to 39%) compared with 67% (95% CI, 55% to 77%) for patients with the wild-type CYP2B6*1 allele in exon 9. Our results suggest that CYP2B6 SNPs influence the efficacy of high-dose Cy and significantly reduce the success of autologous HCT for lymphoma patients with the CYP2B6*5 variant.

MicroRNA-mRNA Pairs Associated with Outcome in AML: From In Vitro Cell-Based Studies to AML Patients.

Cytarabine is the primary chemotherapeutic agent used for treatment of acute myeloid leukemia (AML). Disease relapse after initial remission remains one of the most pressing therapeutic challenges in the treatment of AML. Relapsed disease is often resistant to cytarabine and subsequent salvage therapy is ineffective. Recent studies have shown that some microRNAs (miRNAs) are associated with prognosis, but have not yet explored the role of miRNAs in cellular response to cytarabine. We identified 20 miRNAs that associate with the in vitro cytarabine chemo-sensitivity or apoptotic response of eight AML cell lines. Out of the 20 miRNAs, data on 18 miRNAs was available in AML patients from The Cancer Genome Atlas database. Our stepwise-integrated analyses (step 1 - miRNA-target mRNA that were significantly correlated in AML patients; step 2 - mRNAs from step 1 with significant association with overall survival (OS)) identified 23 unique miRNA-mRNA pairs predictive of OS in AML patients. As expected HOX genes (HOXA9, HOXB7, and HOXA10) were identified to be regulated by miRs as well as predictive of worse OS. Additionally, miR107-Myb, miR-378-granzyme B involved in granzyme signaling and miR10a-MAP4K4 were identified to be predictive of outcome through integrated analysis. Although additional functional validations to establish clinical/pharmacologic importance of miRNA-mRNA pairs are needed, our results from RNA electrophoretic mobility shift assay confirmed binding of miR-10a, miR-378, and miR-107 with their target genes GALNT1, GZMB, and MYB, respectively. Integration of pathogenic and pharmacologically significant miRNAs and miRNA-mRNA relationships identified in our study opens up opportunities for development of targeted/miRNA-directed therapies.

Auranofin induces lethal oxidative and endoplasmic reticulum stress and exerts potent preclinical activity against chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) exhibits high remission rates after initial chemoimmunotherapy, but with relapses with treatment, refractory disease is the most common outcome, especially in CLL with the deletion of chromosome 11q or 17p. In addressing the need of treatments for relapsed disease, we report the identification of an existing U.S. Food and Drug Administration-approved small-molecule drug to repurpose for CLL treatment. Auranofin (Ridaura) is approved for use in treating rheumatoid arthritis, but it exhibited preclinical efficacy in CLL cells. By inhibiting thioredoxin reductase activity and increasing intracellular reactive oxygen species levels, auranofin induced a lethal endoplasmic reticulum stress response in cultured and primary CLL cells. In addition, auranofin displayed synergistic lethality with heme oxygenase-1 and glutamate-cysteine ligase inhibitors against CLL cells. Auranofin overcame apoptosis resistance mediated by protective stromal cells, and it also killed primary CLL cells with deletion of chromosome 11q or 17p. In TCL-1 transgenic mice, an in vivo model of CLL, auranofin treatment markedly reduced tumor cell burden and improved mouse survival. Our results provide a rationale to reposition the approved drug auranofin for clinical evaluation in the therapy of CLL.

Combination of pan-histone deacetylase inhibitor and autophagy inhibitor exerts superior efficacy against triple-negative human breast cancer cells.

Histone deacetylase (HDAC) inhibitors (HDI) induce endoplasmic reticulum (ER) stress and apoptosis, while promoting autophagy, which promotes cancer cell survival when apoptosis is compromised. Here, we determined the in vitro and in vivo activity of the combination of the pan-HDI panobinostat and the autophagy inhibitor chloroquine against human estrogen/progesterone receptor and HER2 (triple)-negative breast cancer (TNBC) cells. Treatment of MB-231 and SUM159PT cells with panobinostat disrupted the hsp90/histone deacetylase 6/HSF1/p97 complex, resulting in the upregulation of hsp. This was accompanied by the induction of enhanced autophagic flux as evidenced by increased expression of LC3B-II and the degradation of the autophagic substrate p62. Treatment with panobinostat also induced the accumulation and colocalization of p62 with LC3B-II in cytosolic foci as evidenced by immunofluorescent confocal microscopy. Inhibition of panobinostat-induced autophagic flux by chloroquine markedly induced the accumulation of polyubiquitylated proteins and p62, caused synergistic cell death of MB-231 and SUM159PT cells, and inhibited mammosphere formation in MB-231 cells, compared with treatment with each agent alone. Finally, in mouse mammary fat pad xenografts of MB-231 cells, a tumor size-dependent induction of heat shock response, ER stress and autophagy were observed. Cotreatment with panobinostat and chloroquine resulted in reduced tumor burden and increased the survival of MB-231 breast cancer xenografts. Collectively, our findings show that cotreatment with an autophagy inhibitor and pan-HDI, for example, chloroquine and panobinostat results in accumulation of toxic polyubiquitylated proteins, exerts superior inhibitory effects on TNBC cell growth, and increases the survival of TNBC xenografts.

Quantum chemical and nuclear magnetic resonance spectral studies on molecular properties and electronic structure of berberine and berberrubine.

The structural and electronic properties of berberine and berberrubine have been studied extensively using density functional theory (DFT) employing B3LYP exchange correlation. The geometries of these molecules have been fully optimized at the B3LYP/6-311G** level. The chemical shift of 1H and 13C resonances in NMR spectra of these molecules have been calculated using the gauge invariant atomic model (GIAO) method as implemented in Gaussian 98. One- and two-dimensional HSQC (1H-13C), HMBC (1H-13C) and ROESY (1H-1H) spectra were recorded at 500 MHz for the berberine molecule in D(2)O solution. All proton and carbon resonances were unambiguously assigned, and inter-proton distances obtained from ten observed NOE contacts. A restrained molecular dynamics (RMD) approach was used to get the optimized solution structure of berberine. The structure of berberine and berberrubine molecules was also obtained using the ROESY data available in literature. Comparison of the calculated NMR chemical shifts with the experimental values revealed that DFT methods produce very good results for both proton and carbon chemical shifts. The importance of the basis sets to the calculated NMR parameters is discussed. It has been found that calculated structure and chemical shifts in the gas phase predicted with B3LYP/6-311G** are in very good agreement with the present experimental data and the measured values reported earlier.